Physical and energetic dissections of enzyme active site properties

Enzymes and the chemical transformations that they catalyze are central to all biological processes. Understanding the physical features of enzymes that enable their exquisite reaction specificities and enormous rate accelerations remains a critical challenge of biochemistry and an important hurdle in rational enzyme design.;Hydrogen bonds form within all enzyme active sites and are key components of enzyme structure and function. How hydrogen bonds facilitate catalysis has been unclear, however, as their properties are difficult to isolate and assess within the complex environment of a folded protein. This thesis focuses on elucidating fundamental physical and energetic properties of enzymatic hydrogen bonds and the nature of the active site environment in which they form.;I utilized nuclear magnetic resonance spectroscopy (NMR) to detect key hydrogen bonds in the active sites of bacterial ketosteroid isomerase (KSI) and photoactive yellow protein (PYP) and determine that these interactions shorten by ∼0.1 A due to electronic rearrangements along the reaction coordinate in both systems. Contrary to expectations from several prior proposals, complementary energetic measurements suggested that hydrogen bond shortening in response to substrate charge rearrangement leads to only a modest increase in the rate of KSI catalysis relative to aqueous solution.;A unique feature of enzymes is their ability to use non-covalent binding interactions to position hydrogen bond donors and acceptors with respect to each other. In the case of KSI, introduction of structural constraints prevented hydrogen bond shortening by up to 0.1 A, resulting in substantially weakened negative charge stabilization within the active site. These results have provided direct insight into the distance scale on which enzymes can distinguish geometric rearrangements and suggest that substantial enzymatic rate acceleration may result from selective energetic stabilization of reaction transitions states based on geometric rearrangements during reactions.;Hydrogen bond networks are ubiquitously present within protein interiors and are proposed to play key roles in biological signal transduction and proton-coupled electron transfer reactions. NMR and deuterium isotope substitution studies of hydrogen bond networks in the KSI and PYP active sites provided direct evidence for robust conformational coupling, with a lengthening of one hydrogen bond by as little as 0.01 A resulting in a shortening of the neighboring hydrogen bond by a similar magnitude. The ability to detect structural rearrangements on this scale provides further evidence that active sites can distinguish subtle geometric differences and suggests that propagation of conformational changes through hydrogen bond networks may play an important role in biological signal transduction.;Structural and biophysical studies have highlighted that the physical environment within an enzyme active site is very different from aqueous solution. Nonetheless, the development of incisive physical probes of the electrostatic environment within proteins has remained challenging. To directly assess the physical nature of the KSI active site and the effects of ligand binding and solvent exclusion on the local electrostatic environment, I incorporated a thiocyanate vibrational probe into KSI. My results suggest that solvent exclusion negligibly alters the electrostatic environment within the KSI active site, contrary to multiple prior proposals. Development of localized negative charge, analogous to the dienolate reaction intermediate, dramatically increases the magnitude of the local electric field due to the charge itself as well as possible ordering of nearby dipoles. These results provide a foundation for future dissections of the electrostatic environment within the active sites of KSI and other enzymes.